Despite dose escalation of cancer chemotherapy and increasingly radical surgery, the overall mortality from ovarian cancer remains unaltered and unacceptable. Therefore there is a definite need for new biologic approaches against this devastating tumor. We have developed a novel method that efficiently enriches for multiple chaperone complexes from tumor lysates using free solution isoelectric focusing. We have documented that chaperone rich cell lysate (CRCL) vaccination is more effective than immunization with purified individual chaperones (heat shock proteins, HSPs). The antigenicity of CRCL can be augmented further by loading them onto dendritic cells (DCs) resulting in protection against murine tumors even in the setting of pre-existing disease. DCs, in the presence of murine tumorderived CRCL, mature and develop superior immunostimulatory capacity when compared to DCs exposed to unfractionated tumor lysate or purified HSPs. We hypothesize that the nature of this enhanced immunogenicity may lie in a broadened range of antigenic peptides that are escorted via the CRCL to DCs, and to the direct activation of DCs by the CRCL. The full range of phenotypic and functional changes that human DCs undergo in response to CRCL remains to be seen and will be studied. We further hypothesize that CRCL pulsed DCs are potent immunostimulants effective in generating tumor specific cytotoxic T lymphocytes (CTL) against ovarian cancer. CRCL pulsed DCs is a promising anti-cancer vaccine that may prove to be useful adjuvant therapy for women suffering from ovarian cancer. To better understand the role of CRCL-DCs as a potential natural immunoaugmentative approach for ovarian cancer, we propose the following specific aims: 1) Generate and biochemically characterize ovarian cancer-derived CRCL. 2) Evaluate the effects of ovarian cancer-derived CRCL on DCs to determine if CRCL induces maturation and/or alters DC function 3) Examine the potential of ovarian cancer-derived CRCL-pulsed DCs to generate tumor specific CTLs. We will isolate CRCL and purified HSPs from ovarian cancers, analyze them by SDS-PAGE and Western blotting for specific chaperone protein content, and assess the yield, stability and endotoxin content of these vaccines. The effects of ovarian cancer CRCL and purified HSPs on blood and ascites derived DCs will then be evaluated. Changes in DC immunophenotype, cytokine production, endocytic/phagocytic activity and immunostimulatory function will be studied. The role of immunosuppressive cytokines found in ascites will be addressed. We will study the potency of ovarian cancer derived CRCL and pure HSPs as antigen sources, the importance of autologous (versus allogeneic) CRCL, the function of blood-derived versus ascites-derived DC in stimulating CTL and the inhibitory effects of cytokines found in i ascites on CTL generation following stimulation by CRCL pulsed DCs.